Abstract
I. Introduction: The McGivney Hemorrhoidal Ligator band (MHL) is an established model of tourniquet induced hind-limb ischemia-reperfusion (IR) injury in rodents. The MHL model has been criticized for its inability to control for confounding factors such as crush injury and neurological damage to the area directly below the rubber band. This problem may be avoided, if complete hind-limb ischemia could be induced with minimal tension. These experiments were designed to (1) define and compare the ex-vivo force generated by the MHL as compared to Orthodontic Rubber Bands (ORB) of different diameters over a 3 hour period; (2) determine whether MHL and ORB create similar grades of in-vivo limb ischemia as measured by Laser Doppler Imaging (3); correlate the tension produced by the MHL and ORB with neurological assessment of murine hind limbs. The overall goal of these experiments is to determine whether orthodontic rubber bands (ORBs) provide an alternate, specific model of complete hind-limb ischemia by generating significantly less force and non-specific neurologic injury than previously established models. II. Methods: Ex- vivo force measurements were performed on 1/8” internal diameter 4.0 oz. ORB and MHL by mounting the bands on circular rods of varying sizes (0.86 and 1.04 cm), secured onto a magnetic-based micromanipulator. Force was then measured using a tensiometer which stretched each mounted band exactly 1 mm. Measurements were done in duplicate and the bands remained on the rods for 90 minutes to evaluate force decay. The in-vivo consequences of limb ischemia documented by applying ORBs and MHL to the hind limbs of anesthetized C57BL6 mice (25-30g, which were then scanned with Laser Doppler Imaging to confirm ischemia for at least 90 minutes. Once complete ischemia was confirmed for 90 minutes, the limbs were reperfused for 24 hours and subjected to neurologic scoring (paralysis, gait) by two blinded observers. III. Results: The force generated by the 4 oz. ORBs and MHL bands were significantly different (MHL: 0.28 + 0.009 vs. ORB: 0.08 + 0.002 kg, p= 0.0079) at 90 minutes. Variations in rod diameter (0.86 vs. 1.04 cm), did not affect the amount of force applied by the 4 oz ORB (p = .5141); In contrast, the force applied by the MHL varied significantly between these two measured diameters (p=0.0248). There was no significant decay in the force applied by the 4.0 oz. ORB up to 90 minutes (p = 0.1862); whereas the MHL showed significant force decay (p=0.0085). Absolute flux measurements compared to baseline perfusion, using LDI showed significant reduction in flow for both the 4 oz. (n = 10, p < 0.0001) ORB and MHL (n = 10, p < 0.0001). Furthermore, the degree of ischemia was equivalent in both ORB and MHL mice-(MHL: 50.1 + 5.6 vs. ORB: 36 + 6.3 flux units, p = 0.1431). At 24 hours reperfusion, the neurological score in MHL mice was significantly worse than ORB mice (MHL: 2.9 + 0.1, ORB: 1.8 + 0.3, p = 0.0137). IV. Conclusion: MHL bands deliver erratic and excessive force to create limb ischemia in rodents. The excessive force may promote non-ischemic neurologic injury, which may be related primarily to crush injury. The ORB provides and attractive alternative to the MHL, and thus may decrease experimental artifacts in studies of rodent hind limb ischemia reperfusion.
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